The acrosome reaction in digitonin-permeabilized sea urchin sperm in the absence of the natural inducer
Por:
Castellano L.E., Lopez-Godinez J., Aldana G., Barrios-Rodiles M., Obregon A., Garcia de De la Torre L., Darszon A., Garcia-Soto J.
Publicada:
1 ene 1995
Resumen:
In many species, the acrosome reaction of sperm is an obligatory step in fertilization. Increases in [Ca2+](i) and pH(i), activation of adenylyl cyclase and inositol trisphosphate generation accompany the egg jelly-induced acrosome reaction of sea urchin sperm. The signaling mechanisms involved are unknown. We used digitonin, a cholesterol-complexing compound, to selectively permeabilize the plasma membrane of sea urchin sperm suspended in a medium that mimics the cytosolic ion composition. Within 6 to 8 min, 30 to 50 ?M digitonin allowed incorporation of the membrane-impermeant dye Hoechst 33258 into the sperm, staining exclusively the nucleus. No alterations in sperm morphology were caused by digitonin at the concentrations used, however, it irreversibly permeabilized the plasma membrane. Permeabilized sperm retained lactate dehydrogenase and actin. When incubated in Ca2+-containing permeabilization buffer (pH 7.8), sperm were capable of undergoing spontaneously the acrosome reaction; this reaction was pH dependent and displayed an absolute Ca2+ requirement. Electron microscopy indicates that the acrosome reaction undergone by permeabilized sperm resembled that induced by egg jelly. Additionally, rhodaminyl-phalloidin staining of sperm reacted under permeabilizing conditions revealed a fluorescent filament in the acrosomal tubule region, demonstrating the occurrence of actin polymerization. Thus, in permeabilized sperm the machinery necessary to perform a [Ca2+](i)- and pH(i)-sensitive acrosome reaction is functionally preserved. Permeabilized sperm offer new avenues to study the molecular bases of the sea urchin sperm acrosome reaction.
Filiaciones:
Castellano L.E.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Lopez-Godinez J.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Aldana G.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Barrios-Rodiles M.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Obregon A.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Garcia de De la Torre L.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Darszon A.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
Garcia-Soto J.:
Inst. Investigacion Biologia Exper., Facultad de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico
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